Human respiratory syncytial virus (HRSV) is the leading viral cause of serious pediatric respiratory tract disease worldwide (Collins, et al., Fields Virology 2:1313-1352, 1996; incorporated herein by reference). RSV outranks all other microbial pathogens as a cause of pneumonia and bronchiolitis in infants under one year of age. Virtually all children are infected by two years of age, and reinfection occurs with appreciable frequency in older children and young adults (Chanock et al., in Viral Infections of Humans, 3rd ed., A. S. Evans, ed., Plenum Press, N.Y., 1989; incorporated herein by reference). RSV is responsible for more than one in five pediatric hospital admissions due to respiratory tract disease, and in the United States alone causes nearly 100,000 hospitalizations and 4,500 deaths yearly. (Heilman, J. Infect. Dis. 161:402-6, 1990; incorporated herein by reference). In addition, there is evidence that serious respiratory tract infection early in life can initiate or exacerbate asthma (Sigurs, et al., Pediatrics 95:500-505, 1995; incorporated herein by reference).
While RSV usually is thought of in the context of the pediatric population, it also is recognized as an important agent of serious disease in the elderly (Falsey et al., J. Infect. Dis. 172:389-394, 1995; incorporated herein by reference). RSV also causes life-threatening disease in certain immunocompromised individuals, such as bone marrow transplant recipients (Fouillard, et al., Bone Marrow Transplant 9:97-100, 1992; incorporated herein by reference).
For treatment of RSV, one chemotherapeutic agent, ribavirin, is available. However, its efficacy and use are controversial. There are also licensed products for RSV intervention which are composed of pooled donor IgG (Groothuis et al., N. Engl. J. Med. 329:1524-1530, 1993; incorporated herein by reference) or a humanized RSV-specific monoclonal antibody. These are administered as passive immunoprophylaxis agents to high-risk individuals. While these products are useful, their high cost and other factors, such as lack of long-term effectiveness, make them inappropriate for widespread use. Other disadvantages include the possibility of transmitting blood-borne viruses and the difficulty and expense in preparation and storage. Moreover, the history of the control of infectious diseases, and especially diseases of viral origin, indicates the primary importance of vaccines.
Despite decades of investigation to develop effective vaccine agents against RSV, no safe and effective vaccine has yet been approved to prevent the severe morbidity and significant mortality associated with RSV infection. Failure to develop successful vaccines relates in part to the fact that small infants have diminished serum and secretory antibody responses to RSV antigens. Thus, these individuals suffer more severe infections from RSV, whereas cumulative immunity appears to protect older children and adults against more serious impacts of the virus.
The mechanisms of immunity in RSV infection have recently come into focus. Secretory antibodies appear to be most important in protecting the upper respiratory tract, whereas high levels of serum antibodies are thought to have a major role in resistance to RSV infection in the lower respiratory tract. RSV-specific cytotoxic T cells, another effector arm of induced immunity, are also important in resolving an RSV infection. However, while this latter effector can be augmented by prior immunization to yield increased resistance to virus challenge, the effect is short-lived. The F and G surface glycoproteins are the two major protective antigens of RSV, and are the only two RSV proteins which have been shown to induce RSV neutralizing antibodies and long term resistance to challenge (Collins et al., Fields Virology, Fields et al. eds., 2:1313-1352, Lippincott-Raven, Philadelphia, 1996; Connors et al., J. Virol. 65(3):1634-1637, 1991; incorporated herein by reference). The third RSV surface protein, SH, did not induce RSV-neutralizing antibodies or significant resistance to RSV challenge.
An obstacle to developing live RSV vaccines is the difficulty in achieving an appropriate balance between attenuation and immunogenicity. Other obstacles include the genetic instability of some attenuated viruses, the relatively poor growth of RSV in cell culture, and the instability of the virus particle. In addition the immunity which is induced by natural infection is not fully protective against subsequent infection. A number of factors probably contribute to this, including the relative inefficiency of the immune system in restricting virus infection on the luminal surface of the respiratory tract, the short-lived nature of local mucosal immunity, rapid and extensive virus replication, reduced immune responses in the young due to immunological immaturity, immunosuppression by transplacentally derived maternal serum antibodies, and certain features of the virus such as a high degree of glycosylation of the G protein. Also, as will be described below, RSV exists as two antigenic subgroups A and B, and immunity against one subgroup is of reduced effectiveness against the other.
Although RSV can reinfect multiple times during life, reinfections usually are reduced in severity due to protective immunity induced by prior infection, and thus immunoprophylaxis is feasible. A live-attenuated RSV vaccine would be administered intranasally to initiate a mild immunizing infection. This has the advantage of simplicity and safety compared to a parenteral route. It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV. It also abrogates the immunosuppressive effects of RSV-specific maternally-derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic complications (Murphy et al., Vaccine 8(5):497-502, 1990; incorporated herein by reference), this has never been observed with a live virus.
A formalin-inactivated virus vaccine was tested against RSV in the mid-1960s, but failed to protect against RSV infection or disease, and in fact exacerbated symptoms during subsequent infection by the virus. (Kim et al., Am. J. Epidemiol., 89:422-434, 1969; Chin et al., Am J. Epidemiol., 89:449-463, 1969; Kapikian et al., Am. J. Epidemiol., 89:405-421, 1969; incorporated herein by reference).
More recently, vaccine development for RSV has focused on attenuated RSV mutants. Friedewald et al., (J. Amer. Med. Assoc. 204:690-694, 1968; incorporated herein by reference) reported a cold passaged mutant of RSV (cpRSV) which appeared to be sufficiently attenuated to be a candidate vaccine. This mutant exhibited a slightly increased efficiency of growth at 26xc2x0 C. compared to its wild-type (wt) parental virus, but its replication was neither temperature sensitive nor significantly cold-adapted. The cold-passaged mutant, however, was attenuated for adults. Although satisfactorily attenuated and immunogenic for infants and children who had been previously infected with RSV (i.e., seropositive individuals), the cpRSV mutant retained a low level virulence for the upper respiratory tract of seronegative infants.
Similarly, Gharpure et al., (J. Virol. 3:414-421, 1969; incorporated herein by reference) reported the isolation of temperature sensitive RSV (tsRSV) mutants which also were promising vaccine candidates. One mutant, ts-1, was evaluated extensively in the laboratory and in volunteers. The mutant produced asymptomatic infection in adult volunteers and conferred resistance to challenge with wild-type virus 45 days after immunization. Again, while seropositive infants and children underwent asymptomatic infection, seronegative infants developed signs of rhinitis and other mild symptoms. Furthermore, instability of the ts phenotype was detected. Although virus exhibiting a partial or complete loss of temperature sensitivity represented a small proportion of virus recoverable from vaccinees, it was not associated with signs of disease other than mild rhinitis.
These and other studies revealed that certain cold-passaged and temperature sensitive RSV strains were underattenuated and caused mild symptoms of disease in some vaccinees, particularly seronegative infants, while others were overattenuated and failed to replicate sufficiently to elicit a protective immune response, (Wright et al., Infect. Immun. 37:397-400, 1982; incorporated herein by reference). Moreover, genetic instability of candidate vaccine mutants has resulted in loss of their temperature sensitive phenotype, further hindering development of effective RSV vaccines. See generally, (Hodes et al., Proc. Soc. Exp. Biol. Med. 145:1158-1164, 1974; McIntosh et al., Pediatr. Res. 8:689-696, 1974; and Belshe et al., J. Med. Virol. 3:101-110, 1978; incorporated herein by reference).
As an alternative to live-attenuated RSV vaccines, investigators have also tested subunit vaccine candidates using purified RSV envelope glycoproteins. The glycoproteins induced resistance to RS virus infection in the lungs of cotton rats, (Walsh et al., J. Infect. Dis. 155:1198-1204, 1987; incorporated herein by reference), but the antibodies had very weak neutralizing activity and immunization of rodents with purified subunit vaccine led to disease potentiation (Murphy et al., Vaccine 8:497-502, 1990; incorporated herein by reference).
Recombinant vaccinia virus vaccines which express the F or G envelope glycoprotein have also been explored. These recombinants express RSV glycoproteins which are indistinguishable from the authentic viral counterpart, and rodents infected intradermally with vaccinia-RSV F and G recombinants developed high levels of specific antibodies that neutralized viral infectivity. Indeed, infection of cotton rats with vaccinia-F recombinants stimulated almost complete resistance to replication of RSV in the lower respiratory tract and significant resistance in the upper tract. (Olmsted et al., Proc. Natl. Acad. Sci. USA 83:7462-7466, 1986; incorporated herein by reference). However, immunization of chimpanzees with vaccinia-F and -G recombinant provided almost no protection against RSV challenge in the upper respiratory tract (Collins et al., Vaccine 8:164-168, 1990; incorporated herein by reference) and inconsistent protection in the lower respiratory tract (Crowe et al., Vaccine 11:1395-1404, 1993; incorporated herein by reference).
Despite these various efforts to develop an effective RSV vaccine, no licensed vaccine has yet been approved for RSV. The unfulfilled promises of prior approaches underscores a need for new strategies to develop RSV vaccines, and in particular methods for manipulating recombinant RSV to incorporate genetic changes that yield new phenotypic properties in viable, attenuated RSV recombinants. However, manipulation of the genomic RNA of RSV and other non-segmented negative-sense RNA viruses has heretofore proven difficult. Major obstacles in this regard include non-infectivity of naked genomic RNA of these viruses and, in the case of RSV, poor viral growth in tissue culture, lengthy replication cycles, virion instability, a complex genome, and a refractory organization of gene products.
Recombinant DNA technology has made it possible to recover infectious non-segmented negative-stranded RNA viruses from cDNA, to genetically manipulate viral clones to construct novel vaccine candidates, and to rapidly evaluate their level of attenuation and phenotypic stability (for reviews, see Conzelmann, J. Gen. Virol. 77:381-389, 1996; Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:11354-11358, 1996; incorporated herein by reference). In this context, recombinant rescue has been reported for infectious respiratory syncytial virus (RSV), parainfluenza virus (PIV), rabies virus (RaV), vesicular stomatitis virus (VSV), measles virus (MeV), rinderpest virus and Sendai virus (SeV) from cDNA-encoded antigenomic RNA in the presence of essential viral proteins (see, e.g., Garcin et al., EMBO J. 14:6087-6094, 1995; Lawson et al., Proc. Natl. Acad. Sci. U.S.A. 92:4477-4481, 1995; Radecke et al., EMBO J. 14:5773-5784, 1995; Schnell et al., EMBO J. 13:4195-4203, 1994; Whelan et al., Proc. Natl. Acad. Sci. U.S.A. 92:8388-8392, 1995; Hoffman et al., J. Virol. 71:4272-4277, 1997; Pecters et al., J. Virol. 73:5001-5009, 1999; Kato et al., Genes to Cells 1:569-579, 1996; Roberts et al., Virology 247(1), 1-6, 1998; Baron et al., J. Virol. 71:1265-1271, 1997; International Publication No. WO 97/06270; U.S. Provisional Patent Application No. 60/007,083, filed Sep. 27, 1995; U.S. patent application Ser. No. 08/720,132, filed Sep. 27, 1996; U.S. Provisional Patent Application No. 60/021,773, filed Jul. 15, 1996; U.S. Provisional Patent Application No. 60/046,141, filed May 9, 1997; U.S. Provisional Patent Application No. 60/047,634, filed May 23, 1997; U.S. Pat. No. 5,993,824, issued Nov. 30, 1999 (corresponding to International Publication No. WO 98/02530); U.S. Provisional Patent Application Serial No. 60/129,006, filed Apr. 13, 1999; Collins, et al., Proc. Nat. Acad. Sci. USA 92:11563-11567,1995; Bukreyev, et al., J. Virol. 70:6634-6641, 1996, Juhasz et al., J. Virol. 71(8):5814-5819, 1997; Durbin et al., Virology 235:323-332, 1997; He et al. Virology 237:249-260, 1997; Baron et al. J. Virol. 71:1265-1271, 1997; Whitehead et al., Virology 247(2):232-239, 1998a; Whitehead et al., J. Virol. 72(5):4467-4471, 1998b; Jin et al. Virology 251:206-214, 1998; and Whitehead et al., J. Virol. 73:(4)3438-3442, 1999, and Bukreyev, et al., Proc. Nat. Acad. Sci. USA 96:2367-2372, 1999, Bucholz et al., J. Virol. 73:251-259, 1999; Collins et al., Virology 259:251-255, 1999, each incorporated herein by reference in its entirety for all purposes).
Based on these developments in recombinant DNA technology, it is now possible to recover infectious RSV from cDNA and to design and implement various genetic manipulations to RSV clones to construct novel vaccine candidates. Thereafter, the level of attenuation and phenotypic stability, among other desired phenotypic characteristics, can be evaluated.
One avenue of investigation toward developing recombinant vaccines has been to engineer viruses to express one or more cytokines or other potential anti-viral molecules. For the most part, these studies involved wild type vaccinia virus and were conducted with the goal of increasing basic understanding of host immunity and viral pathogenesis (see, e.g., Ramshaw et al. Immunol. Rev. 127:157-182, 1992; incorporated herein by reference). The potential utility of cytokine coexpression to improve immune responses for vaccine development has long been contemplated (Ramshaw et al., Trends Biotechol 10:424-426,1992; incorporated herein by reference). However, the use of poxvirus as an object of study reduced the practical application of this concept since smallpox has been eradicated in the human population and the poxvirus vaccine no longer remains in active use.
Examples of these earlier studies investigating the possible utility of cytokine coexpression for vaccine development include a vaccinia virus engineered to express the cytokine interleukin 2 (IL-2). This recombinant virus was reported to be attenuated in immunodeficient athymic nude mice (Flexner et al., Nature 330:259-262,1987; Ramshaw et al., Nature 329:545-546, 1987; incorporated herein by reference). The roles of various immune effectors in clearance and recovery have also been investigated (Karupiah et al., J. Ex. Med. 172:1495-1503,1990; Karupiah et al., J. Immunol. 144:290-298, 1990; Karupiah et al., J. Immunol. 147:4327-4332, 1991; incorporated herein by reference). IL-2 expression by the vaccinia virus recombinant was shown to greatly reduce skin lesions formed by the vaccinia virus in primates, indicating significant attenuation. Despite these preliminary findings, antibody production was determined to be equivalent in the presence, or absence, of IL-2 (Flexner et al., Vaccine 8:17-21, 1990; incorporated herein by reference).
Another example of cytokine coexpression by a recombinant vaccinia virus involved interleukin 4 (IL-4), which was reported to downregulate antiviral cytokine expression and cytotoxic T cell responses, and to exacerbate the viral infection (Sharma et al., J. Virol. 70:7103-7107, 1996; incorporated herein by reference). In yet another study, expression of nitric oxide synthetase by recombinant vaccinia virus was highly attenuating, demonstrating the importance of this host defense mechanism in controlling vaccinia virus infection (Rolph et al., J. Virol. 70:7678-7685, 1996; incorporated herein by reference). Coexpression of IL-5 or IL-6 by a recombinant vaccinia virus resulted in a 4-fold elevation in the level of local IgA (Ramsay et al., Reprod. Fertil. Dev. 6:389-392, 1994; incorporated herein by reference). Coexpression of tumor necrosis factor (TNF) alpha by a recombinant vaccinia virus improved the capacity of mice to control infection, suggesting that this molecule is involved in host defenses against the virus (Sambhi et al., Proc. Natl. Acad. Sci. USA 88:4025-4029, 1991; incorporated herein by reference). Expression of murine IFNxcex3 by a recombinant vaccinia virus greatly reduced virus replication in normal or immune-compromised mice (Kohonen-Corish, Eur. J. Immunol. 20:157-161, 1990; incorporated herein by reference).
These studies have also been extended to retroviruses. Recently, simian immunodeficiency virus (SIV) expressing IL-2 was constructed (Gundlach et al., J. Virol. 71:2225-2232, 1997; incorporated herein by reference). In rhesus monkeys infected with the IL-2-expressing virus the SIV-specific T-cell proliferative response and the antibody titers were similar to those of control virus. SIV-specific CTL were detected in monkeys infected with the IL-2-expressing virus and in two of four control animals. In another study, a SIV recombinant lacking the nef gene and containing the IFNxcex gene was attenuated in vivo, but the cytokine gene was unstable after several weeks of replication in vivo and, in addition, the attenuation was associated with reduced immunogenicity (Giavedoni et al., J. Virol. 71:866-872, 1997; incorporated herein by reference).
Studies with large double-stranded poxviruses, which have approximately 185 ORFs and encode a number of proteins which interfere with host defenses, or with human immunodeficiency virus which also interferes with host defense, are poor models for the nonsegmented negative strand RNA viruses. It has been previously demonstrated that a foreign gene can be expressed from the genome of recombinant respiratory syncytial virus (RSV), and that it is stably maintained (Bukreyev J. Virol. 70: 6634-6641, 1996, incorporated herein by reference). This indicates that it is feasible to coexpress proteins such as cytokines, chemokines, ligands; and other molecules which might alter, increase or otherwise enhance the host response to RSV. The expression of one or more immune modulatory molecules from a recombinant RSV is desirable because it would provide for expression at the local site of RSV antigen production. Furthermore, coexpression obviates the need to separately prepare and administer the immune modulator. However, the strategy of expressing an immune modulator from the genome of a non-retrovirus, i.e., a positive-sense, double-stranded, or negative-sense RNA virus, had not been previously explored.
There is presently a need for augmentation and modification of host immune responses to RSV. This is because an RSV vaccine will be administered to very young infants, an age group which is known to mount immune responses that are less effective and in some respects different than those of adults. For example, neutralizing antibody responses and cytotoxic T cell responses to RSV are reduced in the very young (Kovarik and Siegrist, Immunol. Today 19: 150-152, 1998; Kovarik and Siegrist. Immunol. Cell. Biol. 76:222-236, 1998; Murphy et al., J. Clin. Microbiol. 24:894-989,1986; Risdon et al., Cell. Immunol. 154:14, 1994; incorporated herein by reference). It is generally recognized that the neonatal immune system is biased towards a Th-2 type T helper cell response (i.e., the T helper cell subset defined by IL-4 secretion) (Early and Reen, Eur. J. Immunol. 26:2885-2889, 1996; incorporated herein by reference). T cells from newborns have reduced helper cell activity for B cells, and produce lower quantities of a number of cytokines, including IL-2, interferon gamma (IFNxcex3), and IL-4 (Splawski et al., J. Clin. Invest. 87, 454, 1991; Hassan and Reen, Scand. J. Immunol. 39:597, 1994; Wilson, Pediatr. 54:118, 1991; incorporated herein by reference). Also, young infants typically possess transplacentally-derived RSV-specific IgG, which can interfere with or alter immune responses (Murphy et al., J. Clin. Microbiol. 24:894-989, 1986 and 23:1009-1014, 1986; Siegrist et al., Eur. J. Immunol. 28:4138-4148, 1998; incorporated herein by reference). Finally, certain RSV immunizations can be associated with immunopathologic complications (Murphy et al., Vaccine 8:497-502, 1990; Waris et al., J. Virol. 71:6935-6939, 1997; incorporated herein by reference). Although this typically is not observed with live-attenuated virus infections, there is evidence that individual RSV antigens, and most notably the G protein, have the potential to induce immunopathologic responses even when expressed by live virus (Johnson et al., J. Virol. 72:2871-2880; incorporated herein by reference). These many factors involved in RSV immune responses, immune-mediated protection, and immunopathologic responses indicate a need for developing methods to modulate host responses to an RSV vaccine.
In summary, an urgent need presents itself in the art to develop additional tools and methods to engineer safe and effective vaccines that will alleviate the serious health problems attributable to RSV. In this context, it is necessary to develop a broad and diverse menu of genetic modifications that can be employed, alone or in combination with other types of genetic manipulations, to construct infectious, attenuated RSV vaccine candidates useful for broad vaccine use. Useful manipulations to a live attenuated RSV vaccine virus might include coexpression by a recombinant RSV clone of or more one factors that modulate host immune responses and immune-mediated protection. Surprisingly, the present invention fulfills this need by providing additional tools for constructing infectious, attenuated RSV vaccine candidates.
The present invention provides recombinant RSV (rRSV) which are engineered to express one or more immune modulatory molecule(s). The recombinant virus has a modified genome or antigenome that incorporates a polynucleotide sequence encoding the immune modulatory molecule which is expressed by the virus in infected cells. Preferred immune modulatory molecules for use within the invention are cytokines. However, various other immune modulatory molecules, including chemokines, chemokine or cytokine anatagonists, surface or soluble receptors, adhesion molecules, ligands, and the like, are also useful to alter aspects of viral biology and/or host immune responses to RSV. In more detailed embodiments, the immune modulator is a cytokine selected from an interleukin 2 (IL-2), interleukin 4 (IL-4), interferon gamma (IFNxcex), or granulocyte-macrophage colony stimulating factor (GM-CSF) molecule.
Cytokines and other immune modulatory molecules can be incorporated in a recombinant RSV of the invention in such a manner that they are expressed by the virus in infected cells and modify one or more aspects of viral biology. For example, incorporation and expression of an immune modulator may alter viral infectivity, replication and/or pathogenicity, and may elicit or change one or more host immune responses, for example an anti-RSV neutralizing antibody response, a T-helper cell response, a cytotoxic T cell (CTL) response, and/or a natural killer (NK) cell response.
The invention provides for the intracellular coexpression from a recombinant RSV of a wide variety of proteins found in nature or engineered by recombinant DNA technology. These proteins typically affect hematopoietic cells or, alternatively, can block natural signals and interactions of hematopoietic cells. To construct these recombinant viruses, the viral genome or antigenome is modified to incorporate a polynucleotide sequence encoding the cytokine or other immune modulator molecule(s). The polynucleotide sequences is added or substituted within the genome or antigenome, typically as a separate gene with its own gene start (GS) and gene end (GE) signals. Generally, the polynucleotide sequence encoding the immune modulator is added or substituted into an intergenic or other non-coding region of the recombinant RSV genome or antigenome, at any suitable locus that does not disrupt an open reading frame within the genome or antigenome.
The level of expression of the cytokine or other immune modulator can be adjusted by altering the gene order position of the cytokine-encoding polynucleotide within the recombinant genome or antigenome. For example, the cytokine-encoding polynucleotide can be introduced at any intergenic position or non-coding region within any of the RSV genes. The more upstream or xe2x80x9cpromoter-proximalxe2x80x9d the location of introduction, the higher the level of expression of the modulator will be.
Another method for inserting a gene or genome segment encoding an immune modulatory factor into RSV is to place the cDNA under the control of RSV gene-start and gene-end signals as described above, but to insert the cDNA so that the gene is expressed from the antigenome rather than from the genome. Ideally, the foreign gene is placed immediately downstream from the promoter at the 3xe2x80x2 end of the antigenome, such that this promoter-proximal location ensures a high level of expression.
Yet another method for expressing a cytokine or other immune modulator from RSV to place the ORF for the gene under control of a mammalian internal ribosome entry site, and to insert this ORF into the downstream noncoding region of any one or more of the RSV genes.
Yet another method for expression is by the construction of chimeric or fusion proteins. For example, a protein ectodomain which is desired to be expressed at the surface of the infected cell and virion can be attached to the downstream end of the SH ORF or other non-essential gene, such that the reading frame is undisturbed and a chimeric protein results. In this configuration, the SH moiety provides the signal and membrane anchor, and the C-terminal attached domain is displayed extracellularly.
The expression of one or more immune modulatory molecules by a recombinant RSV is desirable because it provides for expression of the immune modulatory molecule at a local site of RSV antigen production. Thus, coexpression of immune modulatory molecules in accordance with the teachings of the invention obviates the need to separately prepare and administer the immune modulator(s). In addition, recombinant RSV of the invention have other desirable characteristics that are useful for vaccine development. Alteration of the recombinant genome or antigenome to express a cytokine, for example, yields vaccine candidates that exhibit one or more novel characteristics selected from (i) a change in viral growth in cell culture; (ii) a change in viral attenuation in the upper and/or lower respiratory tract of an infected host; (iii) a change in viral plaque size; and/or (vi) a change in immunogenicity, or, alternatively or concomitantly, an ability to elicit an altered host response, e.g., an increased anti-RSV neutralizing antibody response, T-helper cell response, cytotoxic T cell (CTL) response, and/or natural killer (NK) cell response, compared to a host response elicited by wild type or parental (i.e., not expressing cytokine) RSV.
Within preferred aspects of the invention, recombinant RSV express high levels of the introduced cytokine or other immune modulator, for example up to 2.5 micrograms/ml as measured in the medium of infected tissue culture cells. The recombinant viruses are attenuated in vitro and in vivo, yet they exhibit a high level of protective efficacy against wild type RSV in vaccinated subjects are engineered to express undiminished or, more typically, increased levels of viral antigen(s) while also exhibiting an attenuated phenotype. Immunogenic potential is thus preserved due to the undiminished or increased mRNA transcription and antigen expression, while attenuation is achieved through concomitant reductions in RNA replication and virus growth. This novel suite of phenotypic traits is highly desired for vaccine development. Other useful phenotypic changes that are observed in recombinant RSV engineered to express an immune modulator(s) include a change in plaque size and altered cytopathogenicity compared to corresponding wild-type or mutant parental RSV strains.
In combination with the phenotypic effects provided in recombinant RSV which are modified to express a cytokine or other immune modulator, it is often desirable to adjust the attenuation phenotype by introducing additional mutations that increase or decrease attenuation of the recombinant virus. Thus, candidate vaccine strains can be further attenuated by incorporation of at least one, and preferably two or more different attenuating mutations, for example mutations identified from a panel of known, biologically derived mutant RSV strains. Preferred human mutant RSV strains are cold passaged (cp) and/or temperature sensitive (ts) mutants, for example the mutants designated xe2x80x9ccpts RSV 248 (ATCC VR 2450), cpts RSV 248/404 (ATCC VR 2454), cpts RSV 248/955 (ATCC VR 2453), cpts RSV 530 (ATCC VR 2452), cpts RSV 530/1009 (ATCC VR 2451), cpts RSV 530/1030 (ATCC VR 2455), RSV B-1 cp52/2B5 (ATCC VR 2542), and RSV B-1 cp-23 (ATCC VR 2579)xe2x80x9d (each deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., and granted the above identified accession numbers). From this exemplary panel of biologically derived mutants, a large xe2x80x9cmenuxe2x80x9d of attenuating mutations is provided, each of which can be combined with any other mutation(s) within the panel for calibrating the level of attenuation and other desirable phenotypes in recombinant RSV of the invention for vaccine use. Additional mutations which can be thus adopted or transferred to RSV clones modified to express a cytokine or other immune modulator may be identified in various temperature sensitive (ts), cold passaged (cp), small plaque (sp), cold-adapted (ca) or host-range restricted (hr) mutant RSV strains. Additional attenuating mutations may be identified in non-RSV negative stranded RNA viruses and incorporated in RSV mutants of the invention by mapping the mutation to a corresponding, homologous site in the recipient RSV genome or antigenome and mutating the existing sequence in the recipient to the mutant genotype (either by an identical or conservative mutation), as described in U.S. Provisional Patent Application Serial No. 60/129,006, filed Apr. 13, 1999. Additional useful mutations can be determined empirically by mutational analysis using recombinant minigenome systems and infectious virus as described in the references incorporated herein.
Recombinant RSV of the invention selected for vaccine use often have at least two and sometimes three or more attenuating mutations to achieve a satisfactory level of attenuation for broad clinical use. In one embodiment, at least one attenuating mutation occurs in the RSV polymerase gene L (either in the donor or recipient gene) and involves one or more nucleotide substitution(s) specifying an amino acid change in the polymerase protein specifying an attenuation phenotype which may or may not involve a temperature-sensitive (ts) phenotype. Recombinant RSV modified to express a cytokine or other immune modulator may incorporate a ts mutation in any additional RSV gene besides L, for example in the M2 gene. However, preferred vaccine candidates in this context incorporate one or more nucleotide substitutions in the large polymerase gene L resulting in an amino acid change at amino acid Asn43, Cys319, Phe521, Gln831, Met1169, Tyr1321, and/or his1690, as exemplified by the changes, Ile for Asn43, Leu for Phe521, Leu for Gln831, Val for Met1169, and Asn for Tyr1321. Other alternative amino acid changes, particularly conservative changes with respect to identified mutant residues, at these positions can of course be made to yield a similar effect as the identified, mutant substitution. Additional desired mutations for incorporation into recombinant RSV of the invention include attenuating mutations specifying an amino acid substitution at Val267 in the RSV N gene, Glu218 and/or Thr523 in the RSV F gene, and a nucleotide substitution in the gene-start sequence of gene M2. Any combination of one or more of the attenuating mutations identified herein, up to and including a full complement of these mutations, may be incorporated in RSV modified to express an immune modulatory molecule to yield a suitably attenuated recombinant virus for use in selected populations or broad populations of vaccine recipients.
Attenuating mutations may be selected in coding portions of a recombinant RSV genome or antigenome or in non-coding regions such as a cis-regulatory sequence. Exemplary non-coding mutations include single or multiple base changes in a gene start sequence, as exemplified by a single or multiple base substitution in the M2 gene start sequence at nucleotide 7605 (nucleotide 7606 in an exemplary recombinant sequence).
In addition to the above described mutations, infectious RSV modified according to the invention can incorporate heterologous, coding or non-coding nucleotide sequences from any RSV or RSV-like virus, e.g., human, bovine, ovine, murine (pneumonia virus of mice), or avian pneumovirus, or from another enveloped virus, e.g., parainfluenza virus (PIV). Exemplary heterologous sequences include RSV sequences from one human RSV strain combined with sequences from a different human RSV strain in a RSV modified to express a cytokine or other immune modulator. For example, recombinant RSV of the invention may incorporate sequences from two or more wild-type or mutant RSV strains, for example mutant strains selected from cpts RSV 248, cpts 248/404, cpts 248/955, cpts RSV 530, cpts 530/1009, or cpts 530/1030. Alternatively, these novel mutants may incorporate sequences from two or more, wild-type or mutant human RSV subgroups, for example a combination of human RSV subgroup A and subgroup B sequences (see, International Application No. PCT/US/08802 and related U.S. patent applications Ser. Nos. 60/021,773, 60/046,141, 60/047,634, 08/892,403, 09/291,894, each incorporated herein by reference). In yet additional aspects, one or more human RSV coding or non-coding polynucleotides are substituted with a counterpart sequence from a heterologous RSV or non-RSV virus, alone or in combination with one or more selected attenuating mutations, e.g., cp and/or ts mutations, to yield novel attenuated vaccine strains.
In related aspects of the invention, the disclosed modifications relating to the introduction of cytokine encoding sequences are incorporated within chimeric human-bovine RSV, which are recombinantly engineered to incorporate nucleotide sequences from both human and bovine RSV strains to produce an infectious, chimeric virus or subviral particle. Exemplary human-bovine chimeric RSV of the invention incorporate a chimeric RSV genome or antigenome comprising both human and bovine polynucleotide sequences, as well as a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), a large polymerase protein (L), and a RNA polymerase elongation factor. Additional RSV proteins may be included in various combinations to provide a range of infectious subviral particles, up to a complete viral particle or a viral particle containing supernumerary proteins, antigenic determinants or other additional components.
Chimeric human-bovine RSV for use within the invention are generally described in U.S. Patent Application entitled PRODUCTION OF ATTENUATED, HUMAN-BOVINE CHIMERIC RESPIRATORY SYNCYTIAL VIRUS VACCINES, filed by Bucholz et al. on Jun. 23, 2000 and identified by Attorney Docket No. 015280-398100US, and in its priority U.S. Provisional Patent Application Serial No. 60/143,132 (each incorporated herein by reference). These chimeric recombinant RSV include a partial or complete xe2x80x9cbackgroundxe2x80x9d RSV genome or antigenome derived from or patterned after a human or bovine RSV strain or subgroup virus combined with one or more heterologous gene(s) or genome segment(s) of a different RSV strain or subgroup virus to form the human-bovine chimeric RSV genome or antigenome. In certain aspects of the invention, chimeric RSV incorporate a partial or complete bovine RSV background genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a human RSV. In alternate aspects of the invention, RSV modified to express an immune modulatory molecule incorporate a partial or complete human RSV background genome or antigenome combined with one or more heterologous gene(s) or genome segment(s) from a bovine RSV.
Yet additional aspects of the invention involve changing the position of a gene or altering gene order to create or modify a RSV modified to express an immune modulatory molecule. In this context, a number of the foregoing incorporated references have focused on modification of the naturally-occurring order in RSV and other viruses. For example, in RSV the NS 1, NS2, SH and G genes were deleted individually, and the NS1 and NS2 gene were deleted together, thereby shifting the position of each downstream gene relative to the viral promoter. For example, when NS1 and NS2 are deleted together, N is moved from position 3 to position 1, P from position 4 to position 2, and so on. Alternatively, deletion of any other gene within the gene order will affect the position (relative to the promoter) only of those genes which are located further downstream. For example, SH occupies position 6 in wild type virus, and its deletion does not affect M at position 5 (or any other upstream gene) but moves G from position 7 to 6 relative to the promoter. It should be noted that gene deletion also can occur (rarely) in a biologically-derived mutant virus. For example, a subgroup B RSV that had been passaged extensively in cell culture spontaneously deleted the SH and G genes (Karron et al., Proc. Natl. Acad. Sci. USA 94:13961-13966, 1997; incorporated herein by reference). Note that xe2x80x9cupstreamxe2x80x9d and xe2x80x9cdownstreamxe2x80x9d refer to the promoter-proximal and promoter-distal directions, respectively (the promoter is at the 3xe2x80x2 leader end of negative-sense genomic RNA).
Gene order shifting modifications (i.e., positional modifications moving one or more genes to a more promoter-proximal or promoter-distal location in the recombinant viral genome) to create or modify cytokine-expressing RSV of the invention result in viruses with altered biological properties. For example, RSV lacking NS 1, NS2, SH, G, NS1 and NS2 together, or SH and G together; have been shown to be attenuated in vitro, in vivo, or both. It is likely that this phenotype was due primarily to the loss of expression of the specific viral protein. However, the altered gene map also likely contributed to the observed phenotype. This effect is well-illustrated by the SH-deletion virus, which grew more efficiently than wild type in some cell types, probably due to an increase in the efficiency of transcription, replication or both resulting from the gene deletion and resulting change in gene order and possibly genome size. In other viruses, such as RSV in which NS1 and/or NS2 were deleted, altered growth that might have occurred due to the change in gene order likely was obscured by the more dominant phenotype due to the loss of expression of the RSV protein(s).
Yet additional changes will be introduced to change the gene order of cytokine-expressing RSV in an effort to improve the properties of the recombinant virus as a live-attenuated vaccine (see, U.S. Provisional Patent Application Ser. No. 60/213,708 entitled RESPIRATORY SYNCYTIAL VIRUS VACCINES EXPRESSING PROTECTIVE ANTIGENS FROM PROMOTOR-PROXIMAL GENES, filed by Krempl et al., Jun. 23, 2000 incorporated herein by reference). In particular, the G and F genes may be shifted, singly and in tandem, to a more promoter-proximal position relative to their wild-type gene order. These two proteins normally occupy positions 7 (G) and 8 (F) in the RSV gene order (NS1-NS2-N-P-M-SH-G-F-M2-L). In order to increase the possibility of successful recovery, exemplary shifting manipulations have been performed in a version of RSV in which the SH gene had been deleted (Whitehead et al., J. Virol. 73:3438-42 (1999), incorporated herein by reference). This facilitates recovery because this virus makes larger plaques in vitro (Bukreyev et al., J. Virol. 71:8973-82 (1997), incorporated herein by reference). G and F were then moved individually to position 1, or were moved together to positions 1 and 2, respectively. Surprisingly, recombinant RSV were readily recovered in which G or F were moved to position 1, or in which G and F were moved to positions 1 and 2, respectively.
Similarly extensive modifications in gene order for incorporation into cytokine-expressing RSV also have been achieved with two highly attenuated vaccine candidates in which the NS2 gene was deleted on its own, or in which the NS1 and NS2 genes were deleted together. In these two vaccine candidates, the G and F glycoproteins were moved together to positions 1 and 2 respectively, and the G, F and SH glycoproteins were deleted from their original downstream position. Thus, the recovered viruses G1F2xcex94NS2xcex94SH and G1F2/xcex94NS1xcex94NS2xcex94SH had two and three genes deleted respectively in addition to the shift of the G and F genes. To illustrate the extent of the changes involved, the gene orders of wild type RSV (NS1-NS2-N-P-M-SH-G-F-M2-L) and the G1F2/xcex94NS2xcex94SH virus (G-F-NS1-N-P-M-M2-L) or the xcex94NS1xcex94NS2xcex94SH (G-F-N-P-M-M2-L) can be compared. This shows that the positions of most or all of the genes relative to the promoter were changed. Nonetheless, these highly attenuated derivatives retained the capacity to be grown in cell culture.
In other detailed aspects of the invention, recombinant RSVs modified to express an immune modulatory molecule are employed as xe2x80x9cvectorsxe2x80x9d for protective antigens of other pathogens, particularly respiratory tract pathogens such as parainfluenza virus (PIV). For example, recombinant RSV modified to express a cytokine may be engineered which incorporate sequences that encode protective antigens from PIV. The cloning of PIV cDNA and other disclosure supplemental to the instant invention is provided in United States Patent Application entitled PRODUCTION OF PARAINFLUENZA VIRUS VACCINES FROM CLONED NUCLEOTIDE SEQUENCES, filed May 22, 1998, Ser. No. 09/083,793 (corresponding to International Publication No. WO 98/53078) and its priority, provisional application filed May 23, 1997, Ser. No. 60/047,575, U.S. Provisional Patent Application entitled ATTENUATED HUMAN-BOVINE CHIMERIC PARAINFLUENZA VIRUS VACCINES, filed by Bailly et al. on Jul. 9, 1999 and identified by Ser. No. 10/030,951; and U.S. Provisional Patent Application entitled RECOMBINANT PARAINFLUENZA VIRUS VACCINES ATTENUATED BY DELETION OR ABLATION OF A NON-ESSENTIAL GENE, filed by Durbin et al. on Jul. 9, 1999 and identified by Ser. No. 10/030,574; each incorporated herein by reference. This disclosure includes description of the following plasmids that may be employed to produce infectious PIV viral clones or to provide a source of PIV genes or genome segments for use within the invention: p3/7(131) (ATCC 97990); p3/7(131)2G (ATCC 97989); and p218(131) (ATCC 97991); each deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) of 10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A., and granted the above identified accession numbers.
According to this aspect of the invention, recombinant RSVs modified to express an immune modulatory molecule are provided which incorporate at least one PIV sequence, for example a polynucleotide containing sequences from either or both PIV1 and PIV2 or PIV1 and PIV3. Individual genes of RSV may be replaced with counterpart genes from human PIV, such as the F glycoprotein genes of PIV1, PIV2, or PIV3. Alternatively, a selected, heterologous genome segment, such as one encoding a cytoplasmic tail, transmembrane domain or ectodomain of an immunogenic protein may be substituted for a counterpart genome segment in, e.g., the same gene in RSV, within a different gene in RSV, or into a non-coding sequence of the RSV genome or antigenome. In one embodiment, a genome segment from an F gene of HPIV3 is substituted for a counterpart human RSV genome segment to yield constructs encoding chimeric proteins, e.g. fusion proteins having a cytoplasmic tail and/or transmembrane domain of RSV fused to an ectodomain of PIV to yield a novel attenuated virus, and/or a multivalent vaccine immunogenic against both PIV and RSV. Alternatively, one or more PIV3 gene(s) or genome segment(s) can be added to a partial or complete, chimeric or non-chimeric RSV genome or antigenome.
To construct chimeric RSV for use within the invention, heterologous genes may be added or substituted in whole or in part to the background genome or antigenome. In the case of chimeras generated by substitution, a selected gene or genome segment encoding a protein or protein region (e.g., a cytoplasmic tail, transmembrane domain or ectodomain, an epitopic site or region, a binding site or region, an active site or region containing an active site, etc.) from a human or bovine RSV is substituted for a counterpart gene or genome segment in the background RSV genome or antigenome to yield novel recombinants having desired phenotypic changes compared to one or both of the respective wild-type (or mutant parent) RSV strains. As used herein, xe2x80x9ccounterpartxe2x80x9d genes or genome segments refer to counterpart polynucleotides from different RSV sources that encode homologous or equivalent proteins or protein domains, epitopes, or amino acid residues, or which represent homologous or equivalent cis-acting signals which may include but are not limited to species and allelic variants among different RSV subgroups or strains.
In other alternate embodiments, cytokine-expressing RSV are designed as vectors for carrying: heterologous antigenic determinants incorporate one or more antigenic determinants of a non-RSV pathogen, such as a human parainfluenza virus (HPIV). In one exemplary embodiment, one or more HPIV1, HPIV2, or HPIV3 gene(s) or genome segment(s) encoding one or more HN and/or F glycoprotein(s) or antigenic domain(s), fragment(s) or epitope(s) thereof is/are added to or incorporated within the partial or complete HRSV vector genome or antigenome. In more detailed embodiments, a transcription unit comprising an open reading frame (ORF) of an HPIV1, HPIV2, or HPIV3 HN or F gene is added to or incorporated within the chimeric HRSV vector genome or antigenome.
Mutations incorporated within cDNAs, vectors and viral particles of the invention can be introduced individually or in combination into a RSV modified to express a cytokine or other immune modulator, and the phenotypes of rescued virus containing the introduced mutation(s) can be readily determined. In exemplary embodiments, amino acid changes displayed by attenuated, biologically-derived viruses versus a wild-type RSV, for example changes exhibited by cpRSV or tsRSV, are incorporated in combination within a recombinant RSV expressing a cytokine to yield a desired level of attenuation for vaccine use.
The present invention thus provides recombinant RSV modified to express a cytokine or other immune modulator, as well as novel vectors and viral particles which may incorporate multiple, phenotype-specific mutations introduced in selected combinations into the recombinant genome or antigenome to produce a suitably attenuated, infectious virus or subviral particle. This process, coupled with routine phenotypic evaluation, provides recombinant RSV having such desired characteristics as attenuation, temperature sensitivity, altered immunogenicity, cold-adaptation, small plaque size, host range restriction, etc. Mutations thus identified are compiled into a xe2x80x9cmenuxe2x80x9d and introduced in various combinations to calibrate a vaccine virus to a selected level of attenuation, immunogenicity and stability.
In yet additional aspects of the invention, RSVs modified to express a cytokine or other immune modulator, with or without attenuating mutations, are constructed to have additional nucleotide modification(s) to yield a desired phenotypic, structural, or functional change. Typically, the selected nucleotide modification will specify a phenotypic change, for example a change in growth characteristics, attenuation, temperature-sensitivity, cold-adaptation, plaque size, host range restriction, or immunogenicity. Structural changes in this context include introduction or ablation of restriction sites into RSV encoding cDNAs for ease of manipulation and identification.
In preferred embodiments, nucleotide changes within the genome or antigenome of an RSV recombinant expressing a cytokine or other immune modulator include modification of a viral gene by partial or complete deletion of the gene, or reduction or ablation (knock-out) of its expression. Target genes for mutation in this context include genes encoding the attachment (G) protein, fusion (F) protein, small hydrophobic (SH), RNA binding protein (N), phosphoprotein (P), the large polymerase protein (L), transcription elongation factor (M2 ORF1 product), a transcription/translation regulatory protein (M2 ORF2) product, the matrix (M) protein, and two nonstructural proteins, NS1 and NS2. Each of these proteins can be selectively deleted, substituted or rearranged, in whole or in part, alone or in combination with other desired modifications, to achieve novel RSV recombinants.
In one aspect of the invention, an SH, NS 1, NS2, or G gene or M2 ORF2 is modified in a recombinant virus that expresses a cytokine or other immune modulator. For example, each of these genes may be deleted in whole or in part or its expression reduced or ablated (e.g., by introduction of a stop codon or frame shift mutation or alteration of a transcriptional or translational start site) to alter the phenotype of the resultant recombinant clone to improve growth, attenuation, immunogenicity or other desired phenotypic characteristics. For example, deletion of the SH gene in the recombinant genome or antigenome will yield a vaccine candidate having novel phenotypic characteristics such as enhanced growth in vitro and/or attenuation in vivo. In a related aspect, an SH gene deletion, or deletion of another selected non-essential gene or genome segment such as a NS1 or NS2 gene, is constructed in virus modified to express an immune modulator, alone or in combination with one or more different mutations specifying an attenuated phenotype, e.g., a point mutation adopted directly (or in modified form, e.g., by introducing multiple nucleotide changes in a codon specifying the mutation) from a biologically derived attenuated RSV mutant. For example, the SH, NS1, NS2 or M2-2 gene may be deleted in combination with one or more cp and/or ts mutations adopted from cpts248/404, cpts530/1009, cpts530/1030 or another selected mutant RSV strain, to yield a recombinant RSV exhibiting increased yield of virus, enhanced attenuation, improved immunogenicity and genetic resistance to reversion from an attenuated phenotype due to the combined effects of the different mutations.
Alternative nucleotide modifications in recombinant RSVs of the invention can include a deletion, insertion, addition or rearrangement of a cis-acting regulatory sequence for a selected gene in the recombinant genome or antigenome. In one example, a cis-acting regulatory sequence of one RSV gene is changed to correspond to a heterologous regulatory sequence, which may be a counterpart cis-acting regulatory sequence of the same gene in a different RSV or a cis-acting regulatory sequence of a different RSV gene. For example, a gene end signal may be modified by conversion or substitution to a gene end signal of a different gene in the same RSV strain. In other embodiments, the nucleotide modification may comprise an insertion, deletion, substitution, or rearrangement of a translational start site within the recombinant genome or antigenome, e.g., to ablate an alternative translational start site for a selected form of a protein. In one example, the translational start site for a secreted form of the RSV G protein is ablated to modify expression of this form of the G protein and thereby produce desired in vivo effects. In other embodiments, mutations identified by empirical analysis of minireplicons or infectious virus can be incorporated (see, e.g. Kuo et al., J. Virol. 71:4944-4953, 1997; Whitehead et al., J. Virol. 73:3438-3442, 1999; incorporated herein by reference)
In related aspects of the invention, compositions (e.g., isolated polynucleotides and vectors incorporating an RSV-encoding cDNA) and methods are provided for producing an isolated infectious recombinant RSV expressing a cytokine or other immune modulator. Included within these aspects of the invention are novel, isolated polynucleotide molecules and vectors incorporating such molecules that comprise a RSV genome or antigenome which is modified to encode the immune modulator. Also provided is the same or different expression vector comprising one or more isolated polynucleotide molecules encoding N, P, L and RNA polymerase elongation factor proteins. These proteins also can be expressed directly from the genome or antigenome cDNA. The vector(s) is/are preferably expressed or coexpressed in a cell or cell-free lysate, thereby producing an infectious RSV particle or subviral particle.
The above methods and compositions for producing RSV modified to express a cytokine or other immune modulator yield infectious viral or subviral particles, or derivatives thereof. An infectious virus is comparable to the authentic RSV virus particle and is infectious as is. It can directly infect fresh cells. An infectious subviral particle typically is a subcomponent of the virus particle which can initiate an infection under appropriate conditions. For example, a nucleocapsid containing the genomic or antigenomic RNA and the N, P, L and M2(ORF1) proteins is an example of a subviral particle which can initiate an infection if introduced into the cytoplasm of cells. Subviral particles provided within the invention include viral particles which lack one or more protein(s), protein segment(s), or other viral component(s), and are typically infectious.
In other embodiments the invention provides a cell or cell-free lysate containing an expression vector which comprises an isolated polynucleotide molecule encoding an RSV genome or antigenome modified to encode an immune modulator as described above, and an expression vector (the same or different vector) which comprises one or more isolated polynucleotide molecules encoding the N, P, L and RNA polymerase elongation factor proteins of RSV. One or more of these proteins also can be expressed from the genome or antigenome cDNA. Upon expression the genome or antigenome and N, P, L, and RNA polymerase elongation factor proteins combine to produce an infectious RSV viral or subviral particle.
The recombinant RSVs of the invention are useful in various compositions to generate a desired immune response against RSV in a host susceptible to RSV infection. Attenuated RSVs of the invention are capable of eliciting a protective immune response in an infected human host, yet are sufficiently attenuated so as to not cause unacceptable symptoms of severe respiratory disease in the immunized host. The attenuated virus or subviral particle may be present in a cell culture supernatant, isolated from the culture, or partially or completely purified. The virus may also be lyophilized, and can be combined with a variety of other components for storage or delivery to a host, as desired.
The invention further provides novel vaccines comprising a physiologically acceptable carrier and/or adjuvant and an isolated RSV particle or subviral particle modified to express a cytokine. In preferred embodiments, the vaccine is comprised of a mutant RSV having a genome or antigenome modified to encode a cytokine and having at least one, and preferably two or more attenuating mutations or other nucleotide modifications as described above to achieve a suitable balance of attenuation and immunogenicity. The vaccine can be formulated in a dose of 103 to 106 PFU or more of attenuated virus. The vaccine virus may elicit an immune response against a single RSV strain or antigenic subgroup, e.g. A or B, or against multiple RSV strains or subgroups. In this regard, recombinant RSV of the invention can be combined in vaccine formulations with other RSV vaccine strains or subgroups having different immunogenic characteristics for more effective protection against one or multiple RSV strains or subgroups.
In related aspects, the invention provides a method for stimulating the immune system of an individual to elicit an immune response against RSV in a mammalian subject. The method comprises administering a formulation of an immunologically sufficient amount of an attenuated RSV modified to express a cytokine or other immune modulator, in a physiologically acceptable carrier and/or adjuvant. In one embodiment, the immunogenic composition is a vaccine comprised of an isolated RSV particle or subviral particle modified to express a cytokine and having at least one, and preferably two or more attenuating mutations or other nucleotide modifications specifying a desired phenotype as described above. The vaccine can be formulated in a dose of 103 to 106 PFU or more of attenuated virus. The vaccine may elicit an immune response against a single RSV strain or antigenic subgroup, e.g. A or B, or against multiple RSV strains or subgroups. RSV recombinants of the invention can be combined with RSV having different immunogenic characteristics in a vaccine mixture, or administered separately in a coordinated treatment protocol, to elicit more effective protection against one RSV strain, or against multiple RSV strains or subgroups. Preferably the immunogenic composition is administered to the upper respiratory tract, e.g., by spray, droplet or aerosol. Often, the composition will be administered to an individual seronegative for antibodies to RSV or possessing transplacentally acquired maternal antibodies to RSV.